RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain

Abstract

AbstracthnRNPA1 is a member of heteronuclear ribonucleoproteins that has been shown to promote telomere elongation apart from its roles in RNA transport and alternative splicing. It is a modular protein with an N-terminal domain called UP1 that consists of two RNA Recognition Motifs (RRM1 and RRM2 domains) and a C-terminal region that harbors functional motifs such as RGG-box, a prion-like domain, and a nuclear shuttling sequence. UP1 has been reported to bind and destabilize telomeric DNA G-quadruplexes and thereby participate in DNA telomere remodeling. An RGG-box motif that consists of four RGG repeats (containing arginine and glycine residues) is located C-terminal to the UP1 domain and constitutes an additional nucleic acid and protein-binding domain. However, the precise role of the RGG-box of hnRNPA1 in telomere DNA recognition and G-quadruplex DNA unfolding remains unexplored. Here, we show that the isolated RGG-box interacts specifically with the structured telomere G-quadruplex DNA but not with the single-stranded DNA. Further the interaction of the RGG-box with the G-quadruplex DNA is dependent on the loop nucleotides of the G-quadruplex. Finally, we show that the RGG-box enhances the G-quadruplex unfolding activity of the adjacent UP1 domain. We propose that UP1 and RGG-box act synergistically to achieve complete telomere G-quadruplex DNA unfolding.

High Affinity binding of Yeast Nucleolin Nsr1 to Co-transcriptionally Formed G4 DNA Obstructs Replication and Elevates Genome Instability

“…For the complementation study, a full length NSR1 gene construct along with its own promoter was cloned into the centromeric plasmid pRS316. Since the C-terminal RGG domain of Nucleolin has been shown to be important for inducing and stably binding G4 structures [40,44], we also cloned the N-terminal 1-350 residues of Nsr1 lacking the RGG domain (Nsr1ΔRGG) ( Figure 3A). The expression of both full length Nsr1 and Nsr1ΔRGG was confirmed using anti-Nsr1 antibody ( Figure S2A).…”

“…Other proteins with verified roles in DNA metabolism and cancer development that contain RGG motifs include Mre11 (a DSB processing enzyme), Mll4 (a histone methyltransferase), and Ews (Ewings Sarcoma protein; DNA damage response protein). Recently, the RGG-box of hnRNPA1, a member of ribonucleoproteins, was shown to bind specifically to the telomeric G4 DNA [44]. Earlier biochemical analysis showed that the RGG domain of hNCL alone can bind G4 DNA with high affinity [45].…”

“…Once bound to the G4 DNA, NCL can not only act as molecular chaperone for the G4 folding but also self-associate to create a higher order complex [65]. The self-association function of NCL, as well as interaction with other proteins, is dependent on the C-terminal domain rich in RGG residues [40,44]. Interestingly, when overexpressed from a centromeric plasmid, Nsr1 protein lacking the RGG domain (Nsr1∆RGG) did not elevate but rather slightly reduced the rate of recombination at the pTET-lys2-GTOP cassette in a top1∆ background ( Figure 4B).…”

“…The binding of NCL to the G4-forming hexanucleotide repeat expansion (HRE) (GGGGCC)n in C9orf72 has been reported to be responsible for the initiation of molecular cascades that lead to neurodegenerative diseases [17]. NCL is composed of three main structural domains; the amino-terminal domain containing four acidic stretches was shown to induce chromatin decondensation through interaction with histone H1 [43], while the central region containing tandem RNA binding domains (RBDs) and the multiple RGG (arginine/glycine/glycine) boxes at the C-terminal domain contribute to its high-affinity interaction with G4 DNA [44,45].…”

Combining 1,3‐Ditriazolylbenzene and Quinoline to Discover a New G‐Quadruplex‐Interactive Small Molecule Active against Cancer Stem‐Like Cells

“…The G-quartets can be linked by interveningl oop sequences so that they can stack on top of each other to form aG 4, which is additionally stabi-lized in solutionb ym onovalent cations coordinated witht he O6 atoms of guanines. [1] There is now good evidence that G4s can be formed in cells, [4,5] where they have important regulatory roles such as in telomerem aintenance, [6] DNA replication, epigenetic responses, and control of gene expression. [1,7,8] Moreover,t he enrichment of theses tructures in promoter regions of, in particular, proto-oncogenesh as been revealed by informaticsa nalysiso ft he human genome [9] and supported by antibody-based G4 chromatin immunoprecipitation and highthroughput sequencing.…”